Studies on the binding of S-1 to regulated actin suggest that the troponintropomyosin-actin complex exists in two forms, a weak and a strong S-1 binding form, with Ca-2+ and S-1 cooperatively shifting the equilibrium in the direction of the strong form. We have now examined this cooperativity in S-1 binding by using a fluorescent probe which is located on the troponin-I subunit of the troponin-tropomyosin complex. Both in the presence and absence of Ca-2+, S-1 induces a fluorescence change in this modified regulated actin and this fluorescence change occurs at a much lower S-1 concentration than is needed to saturate the actin filament with S-1. More importantly, this fluorescence change appears to be correlated with the transition of the troponin-tropomyosin-actin complex from the weak to the strong binding form. Therefore, the fluorescence data support our binding data which suggest that S-1 binds cooperatively to regulated actin both in the presence and absence of Ca-2+. In addition, we have shown that under conditions where S-1 binds noncooperatively to regulated actin, the S-1 induces no change in fluorescence. This experiment was done by measuring the fluorescence induced by the binding of the stable S-1.ATP analog, pPDM S-1, in the presence of PPi or ATP and the absence of Ca-2+. As predicted by our model, the binding of pPDM S-1 to regulated actin does not shift the troponin-tropomyosin-actin complex from the weak to the strong form, because the pPDM S-1 does not bind cooperatively to regulated actin.